Apparent cortisone reductase deficiency (ACRD) is a human disease in which the metabolic clearance of cortisol is increased, leading to over activity of the adrenal cortex, increased secretion of adrenal androgens, and a phenotype similar to polycystic ovary syndrome (PCOS)/metabolic syndrome/syndrome X. Our previous work has shown that ACRD is a digenic disease, requiring carriage of mutations in two genes, HSD11B1 (encoding the type 1 or liver isozyme of 11beta-hydroxysteroid dehydrogenase) and H6PD (encoding hexose-6-phosphate dehydrogenase). We propose to elucidate the mechanisms by which these mutations interact to cause disease. We will define the expression of H6PD in a variety of murine and human tissues by measuring mRNA levels using quantitative PCR, and by measuring protein expression using immunoblotting and assays of enzymatic activity. We will investigate the direct role of H6PD in determining the set point of 11beta-HSD1 activity by expressing H6PD and 11beta-HSD1 in bacteria, and by analyzing the effect of varying H6PDH expression upon 11beta-HSD1 oxo-reductase activity in mammalian cells. We will determine functional consequences of variations in H6PD activity upon phenotype of primary human adipocytes and hepatocytes. We will develop a mouse model of ACRD. To do this, we will produce a mouse with a targeted inactivation of H6PD, and cross this to a mouse with an inactivated HSD11B1 allele. We will breed double heterozygous mutant mice to produce a mouse model of ACRD. Phenotypic characterization will focus on gene and protein expression, 11a-HSD1 activity and equilibrium set-point in liver and adipose tissue, development of the adrenal cortex and levels of expression for key genes regulating steroidogenesis, assays of adrenal function, adipose tissue development, and hepatic enzyme expression. Finally, we will genotype women with polycystic ovary syndrome to determine the degree to which polymorphisms in the human HSD11B1 and H6PDH genes are risk factors for the development of this condition. Subsets of the genotyping data will be analyzed as an association study, as an affected sib pair study, and by transmission disequilibrium testing. Additional polymorphisms in these genes will be sought in PCOS subjects.